1. Biosensors
Recently developed microfabrication techniques have facilitated the specific design and preparation of diagnostic microdevices that integrate the recognition properties of biological macromolecules with the sensitivity of electrochemical transducers. These transducers consist of conductive microsurfaces, which when modified by appropriate ligands, enable the detection and measurement of biological interactions occurring thereupon. Particularly attractive as microdevice transducers are microelectrodes covered by electropolymerized conductive polymers (ECPs) capable of immobilizing various biomolecules and ligands such as enzymes, proteins, antibodies and small ligands (for example, catalysts, porphyrins, DNA/cDNA/RNA sequences and the like). ECPs are infusible and insoluble organic materials which show intrinsic electronic conductivities due to their highly conjugated backbones.
The main advantages of ECPs are (1) a capacity to be mildly electrogenerated on microelectrodes of defined geometries, (2) electroconductivity, allowing strictly controlled growth of the polymeric layers, (3) the relative ease of chemically modifying the electropolymerizable precursor monomers, and (4) the known compatibility of the oxidative/reductive electrochemical conditions with peptides and DNA sequences for film generation onto electrodes.
Basically, in order to build biosensing microdevices successfully, two main strategies can be envisaged for stable immobilization of any biomolecule or ligand onto an electrode via ECPs: entrapment within a growing polymer during the respective electrochemical growth process or covalent attachment onto a preformed functionalized polymer. In the case of proteins and for some smaller ligands, the former strategy is plagued by a number of problems: difficult access of analytes to the immobilized proteins and reduced activities of entrapped enzymes. Additionally, it should be noted that the direct grafting of precursor monomer-linked biological macromolecules does not provide homopolymerization although an electrochemical copolymerization process with unsubstituted monomers can afford the desired biopolymers. Consequently, much of the effort to develop ECPs as matrices for the covalent grafting of various biomolecules or ligands (e.g., proteins, antibodies) in the biosensor field has been directed towards the search for polymers with improved performance chemically activated surfaces. For example, useful precursor polymers have been produced by polymerizing specially functionalized racemic thiophene or pyrrole monomers onto metallic microelectrodes. Additionally, the great versatility of ECPs find a novel and exciting application in the newly emerging field of biochip technologies and related surface modifications.
2. The Biochip Field
Shifting from single analyte tests (biosensors) to integrated assemblies, or arrays of molecular probes allowing for massive parallel throughput screening, has fueled the active development of ligands or molecular probes matrices (biochips) functionalized by diverse DNA, cDNA, peptides or proteins entities. Highly parallel screening of suitable analytes at a high density and capacity format are the main characteristics of biochips technologies. But, functionalizing a given support surface as a bidimensional matrix of molecular probes without cross-contamination requires, developing a versatile surface chemistry able to selectively link these molecular probes onto the support, preferably covalently for analytical reliability. Different kinds of surface chemistries have been designed and developed in this field owing (1) to diagnostic applications, (2) to desired surface densities of molecular probes, (3) to their chemical types, as well as (4) to detection methods for fingerprint analysis after analyte incubation (using, for example, radioactivity, fluorescence, luminescence, electroluminescence and electrochemical techniques). For example, the Affymetrix (Santa Clara, Calif., U.S.A) method consists of arrays of target DNA sequences synthesized combinatorially on aminated silicon wafers, using protected photosensitive N-acyl-deoxynucleoside phosphoramidites, for the consecutive elongation of the oligodeoxynucleotide probes on the chip surface. High density DNA biochips have been produced (400,000 DNA probes) onto a 1.28 cm2 silicon surface. Moreover, the APEX silicon chip of Nanogen (San Diego, Calif., U.S.A.) contains an array of 64 micro-electrodes, each with a different DNA segment attached via an aminated or streptavidin permeation polymeric layer. Electric currents are used to direct the oligodeoxynucleotide probes onto the activated electrodes with a concomitant increase in the rapidity of the hybridization reaction.
Regarding the use of ECPs, another method makes use of functional DNA-linked ECPs on a chip bearing 128 microelectrodes (CisBio International, Bagnols-sur-Ceze, France). The microelectrodes are sequentially functionalized by oxidative electropolymerization of oligodeoxynucleotide-pyrrole probes. Those chips have been used for genetic testing and pathogen identification based on DNA sequences probes.
In the same trend of research and mainly driven by cost and technical simplicity, the present inventor and associates have developed a novel and general concept for the production of DNA/cDNA/RNA/proteins biochips which use biotinylated surfaces and the well-known avidin-biotin system for quasi-covalent probe attachment onto the biochips surfaces (Kd=10−15). This substrate-protein interaction is always viewed as an extraordinary tool for bioconjugation in molecular biology and numerous reagents have been chemically modified by biotin for diagnostic purposes like immunological systems. Interestingly, this approach to diagnostic biochips integrates surface modifications by a sublayer of biotinylated-poly(dipyrrole) ECPs with the use of commercially available micro/nanospotting devices for surface arraying to produce the required matrices of probes.
3. Functional ECPs
U.S. Pat. No. 6,197,881 describes electrically conductive copolymers suitable for presenting a wide variety of biologically interesting molecules on a surface, especially in a surface array or matrix useful in the preparation of biochips and biosensors. More specifically, there is taught a conductive copolymer consisting of two oxidizable and polymerizable monomers “A” and “B”. A is a first polymerizable monomer containing a biotin (or a complex of biotin) as a functional group and it produces an electrically conductive polymer when polymerized. B is a second polymerizable monomer, lacks the biotin functional group, but may contain a group having a desired chemical functionality, which when copolymerized with monomer, or polymer A produces an electrically conductive copolymer. A and B are independently selected from the group consisiting of pyrrole, carbazole, acetylene, azine, p-phenylene, p-phenylene vinylene, pyrene, thiophene, furan, selenophene, pyridazine, aniline and tyramide.
The oxidizable pyrrole moiety of the A and B monomers described in U.S. Pat. No. 6,197,881 form, after oxidation, long polymeric chains (see below the indicated polymer growth directions). The resulting copolymer films exhibit a highly cross-linked skeleton and, hence, the mechanical stability and ionic permeability essential for polymer growth.
wherein X represents OH, —HN—(CH2)n—NH2 or —HN—(CH2)n—NH-biotin; n represents an integer equal or greater than 1; and n2 and m2 indicate the degree of polymerization of the drawn polymer and are such that averaged molecular weights of more than 1,000,000 Daltons are routinely obtained.
The proximity of an electron withdrawing ester group to one of the two oxidizable groups should differentiate them oxidatively, and enable us to modulate the structural and physico-chemical properties of resulting films.
More specifically, the conductive copolymer of the U.S. Pat. No. 6,197,881 is formed by electropolymerization of two co-monomers, only one of them containing a functional group capable of binding a ligand, having the general structure:

A and B are oxidizable moeities (pyrrole; carbazole, thiophene and the like), A is chemically functionalized whereas B is not functionalized. A and B can represent identical or non-identical moieties
Fg is a functional group capable of binding a ligand and it is linked to the oxidizable moiety A. It is biotin (or a biotin-containing complex) in the monomers described in the U.S. Pat. No. 6,197,881;
The linker separates the oxidizable moeity A from the functional group Fg. The linker can be of any chemical nature;
n1, m1, n2 and m2 represent the number of the individual monomers in the copolymer and they may be identical, or non-identical;
d is the average distance between two Fg groups linked to oxidized A units in the copolymeric chain. This distance is absolutely not homogenous in the case of co-polymers.
U.S. Pat. No. 6,197,881 provides some preliminary data on biochip preparation using biotinylated ECPs.
There are some major disadvantages characterizing the polymers of the U.S. Pat. No. 6,197,881:
1. The copolymerized product described in U.S. Pat. No. 6,197,881 contains functional groups in a non-organized arrangement in which the distance between adjacent functional groups, each capable of binding a ligand, is irregular and sporadic.
2. Of the two electropolymerizable monomers participating in the formation of the copolymer of U.S. Pat. No. 6,197,881, only one contains a functional binding group (biotin). The functional groups (biotin) of such copolymer have the limitation of sporadically spreading over the biochip surface. Consequently, it is neither possible to plan the desired distance between two adjacent active combining groups nor it possible to surface engineer tailor-made nanoscale assemblies in which the distance between the adjacent active binding groups is pre-determined in rational way.
3. Neither of the monomers described in U.S. Pat. No. 6,197,881 contains optical active chiral center. Consequently, the polymer layers lack the stereoselectivity frequently required for specific binding of a ligand.
In light of the above, it is clear that there is a need for a new polymerized conductive polymer (ECP) for surface engineering of novel nanoscaled assemblies. These assemblies are useful in a wide scope of biological applications relying mainly on the covalent grafting of specific ligands (such as, for example, proteins; DNA/cDNA/RNA sequences) to active binding groups exist on spherical, hemispherical or planar polymeric surfaces structures at A nanoscale level.
More specifically, there is a need for:
(a) a C2-C6 fully symmetrical polymerizable monomer containing at least two identical oxidizable groups and at least two chiral centers, wherein each of the chiral carbon atom links a carboxylated group capable of binding a functional group or a ligand; and
(b) a polymerizable monomer containing at least two non-identical oxidizable groups and at least two chiral centers, wherein each of the chiral carbon atoms links a carboxylated group capable of binding a functional group or a ligand.
Such a group of monomers is henceforth referred to as C2-C6 “pseudo-symmetrical” monomers.
There is also a need for C2, C3, C4, C5, and C6 symmetrical and pseudo-symmetrical monomers, as described above, wherein during the (co)polymerization process the oxidizable groups are subjected to diverse electrochemical and chemical oxidation/reduction techniques.
There is also a need for polymeric chiral-linked functional groups (ECP) adsorbed on microelectrodes that can be used for the covalent grafting of biological ligands such as proteins, enzymes; antibodies amino-linked DNA/cDNA/RNA.
There is also a need for an array of biosensors of nanoscaled electrodes capable of chirally matched-mismatched grafting of chiral biological probes onto chiral polymeric films.
There is also a need for highly reticulated polymeric (ECP)-, and/or composite polymeric (ECP)-magnetic nanoparticles for covalently grafting various biological probes.
There is also a need for the epitaxial growth of chiral monomers onto oxidizable siloxane-based monolayers as a novel way to engineer conductive surfaces.
There is also a need for array of composite gold-carboxylated polymeric ECPs nanoelectrodes.
There is also a need for a nanoscaled chiral polymeric (ECP) cylinder capable of covalently grafting of amino-containing biological probes.
There is also a need for biosensor and/or biochip devices comprising nanoscaled chiral polymers (ECP) capable of covalently grafting amino-containing biological probes.